Progress 04/15/23 to 04/14/24
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We sent and are sending agraduate student (Alicia Denton) to the midwest meeting of the American Society of Animal Science (ASAS) (Madison, WI; March 2024)and the national meeting of the ASAS (Calgary, Alberta, Canada; July 2024) this summer. She will present the abstract entitiled "Creation of gene-edited pigs for functional analysis of growth hormone receptor promoters in swine"at the national meeting this summer. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We plan to complete studies 2A and 2C. For Study 2A, experimental animals will be weighed within 24 h of birth and weekly until weaning. From weaning, pigs will be weighed once every two weeks. At each weighing, crown-rump length, chest circumference, and height at the shoulders will be measured and used to calculate the ponderal index. A circumference of the lower front leg (radius and ulna) will be measured at each weighing to estimate bone size. Blood will be collected by jugular venipuncture at weaning and once every four weeks thereafter. Plasma will be analyzed to measure GH andIGF1. At each weighing, backfat depth (BF) and loin eye area (LEA) will be measured at the dorsal midline at the 10th rib using an Aloka 500V real-time ultrasound machine fitted with a 3.5 MHz linear transducer. All gilts will be exposed to boars for ten minutes daily beginning at 130 d of age to determine age at puberty. Vulva scores will be assigned to each gilt daily to track development. Blood samples will be assayed for progesterone to confirm the age of puberty. Scrotal height and width will be recorded weekly on each boar beginning at 130 d of age and used to estimate testicular volume. Each boar will be exposed to a collection dummy once weekly by a technician experienced in training boars and collecting semen. All collected samples will be evaluated for sperm concentration and morphology to estimate puberty.For Study 2C,we will slaughter all of the study animals for collection of tissues (approximately 4 males and 4 females for -/-, -/+, and +/+ for the GHR P1 KO and approximately 4 males and 4 females for -/-, -/+, and +/+ for the GHR P2 KO).A sample of abdominal adipose tissue, longissimus dorsi muscle, semitendinosus muscle, and liver will be collected and flash frozen in liquid nitrogen for RNA extraction. We will measure the weight of all major organs systems (heart, lung, liver, intestine, reproductive, etc.) and calculate a percentage of body weight for the respective tissues. Following a 24 h chilling, carcasses will be broken into primal cuts. Each primal cut will be weighed and dissected into fat, lean and bone to estimate composition and distribution of body tissues.Total RNA will be extracted and quantitative real-time RTPCR will be used to assay for the amount of cDNA (i.e., mRNA) in each sample usingspecific PCR primers for GHR 1A, GHR 1B, GHR 1C, and IGF1. The amount of each mRNA (relative to the control sample) will be calculated.
Impacts
What was accomplished under these goals?
We have created 4 genetic lines of GHR promoter knock out (KO) pigs (two lines of GHRP1 KO and two lines of GHRP2 KO). Tissues from pigs have been collected and the promoter KO lines have been tested to verify that the promoter KO leads to the expected changes of GHR mRNA expression in liver and muscle. These milestones (creation of two genetic lines) represent the completion of Objective 1 from the original proposal.We are currently breeding the heterozygous pigs (+/-) to create litters of +/+, +/-, and -/- pigs for study (Objective 2). We are currently conducting studies 2A and 2C from Objective 2.Piglets from theGHRP1line were +/+ (n=3), +/- (n=10), and -/- (n=2) across two litters. Birthweights (1.5±0.1, 1.4±0.1, and 1.5±0.2 kg, respectively) and body weight (BW) at 2 mo of age (32.7±2.9, 27.0±1.6, and 27.0±3.5 kg, respectively) were similar (P>0.10) forGHRP1piglets. Piglets from theGHRP2line were +/+ (n=2), +/- (n=6), and -/- (n=9) across two litters. Birthweights were similar (1.4±0.3, 1.3±0.1, and 1.0±0.1 kg, respectively) butGHRP2-/- pigs weighed less (P<0.01) at weaning compared withGHRP2+/+ orGHRP2+/- piglets (6.4±0.8, 6.5±0.4, and 4.5±0.4 kg, respectively). Monthly BW for theGHRP2-/- piglets were less (P<0.001) when compared withGHRP2+/+ orGHRP2+/- piglets. At 5 months of age, theGHRP2-/- piglets were approximately 50% of the BW ofGHRP2+/+ orGHRP2+/- (89.6±6.7, 89.6±3.9, and 43.1±3.2 kg, respectively). The deletion ofGHRP2(andGHR1BmRNA) had a profound effect on pig growth. TheGHRP1deletion (GHR1AmRNA) did not affect growth with the caveat that theGHRP1litters had not reached maturity. We currently have 3 additional +/- x +/- GHRP2 and 2 additional +/- x +/- GHR P1 matings to farrow this spring. There are a total of 19 pigs on growth trials, 11 pigs that are either pregnant or being bred (+/- x +/- crosses) and 6 pigs (boars) that are held in a facility with strict biosecurity (for recreation of the lines, if necessary). In separate work we have found that at GHRP2 -/- x -/- cross is fertile (two litters produced from this cross. This indicates that the GHR P2 is not required for pregnancy.
Publications
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Progress 04/15/22 to 04/14/23
Outputs
Target Audience:
Nothing Reported
Changes/Problems:We have encountered two major problems. 1. The COVID-19 pandemic slowed the bench work needed to complete this project. Many of the laboratory supply chains needed to do the proposed work including the supply of ovaries that are used for collection of oocytes for somatic cell nuclear transfer (SCNT) were disrupted. 2. The GHR promoter regions have proven to be difficult regions of the genome to knockout. Many rounds of transfections were required to generate the cells lines used for SCNT. Most of the successul KO were "hets" meaning that only one of the alleles were knocked out. This meant that we did not have biallelic knockouts to start our breeding programs. Regardless, we now have a large number of pigs "on the ground" and we can mate the pigs to produce litters of KO and control pigs. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Long-term goal: Regardless of line, mate +/- female to +/- male to produce litters of +/+, +/-, -/- pigs. Use +/+ as the control. Second option: mate -/- female to +/- male (or visa-vera) to produce litters of +/- and -/- pigs. Use the +/- as the control. We think the +/- could be as good as the +/+. This mating we give us a large number of KO pigs. GHR P2 KO pigs 131-1 (GHRP2 +/-) clone sow will be bred to 37-5 (GHRP2 +/-) or 37-8 (GHRP2 +/-) to produce GHR P2 +/+, +/-, -/- litter. We may treat with Matrix and(or) PG600 to get her cycling (she appears to be non-cyclic at this time). 162-1 (GHR P2 -/-) clone boar. This pig is 378 days old (b 3-8-2022). We plan to use this pig and mate with GHR +/- gilts to produce litters of GHR P2 +/- and -/- piglets. 37-4 (GHR P2 +/-) gilt wast bred to 37-8 (GHR P2 +/-) boar on Jan 30, 2023 to produce GHRP2 +/+, +/-, -/- litter. This 37-4 pig is pregnant and is due May 24, 2023. We will make measurements on this litter. 37-5 (GHR P2 +/-) 37-8 (GHR P2 +/-) boars. We will use these boars to generate addtional litters for study. 126-3 (GHRP2 +/- boar). We will use thisboarto generate addtional litters for study. Complex gilts bred to 162-1 (GHR P2 -/-) clone boar (DJ). Should return to estrus this week or confirmed pregnant next week. Returns to estrus will be bred back to 162-1. We plan to use the progeny of this making (GHR P2 +/-) to generate addtional pigs for study. GHR P1 lines: There are two KO alleles (GHRP1 long and GHRP1 short) 123-1 and 123-3 (GHR P1 +/-; short allele) clone boars. These pigs are 110 days old (b 12-1-2022). Will eventually breed to P316, P320, and P323 (GHR P1 -/-; short/long allele) clone sows to produce litters of GHR P1 +/- and -/- piglets. P316, P320, P323 (GHRP1 -/-; short/long allele) clone sows. These pigs are 320 days old (b 4-5-2022). Farrowed 2-18-2023 (P316), 2-19-2023 (P323) and 2-20-2023 (P320). Weaned 3-16-2023. Will breed back to WT boar (different from the boar used for the first litter) to produce +/- piglets. P316, P320, and P323 piglets. Will keep 3GHR P1 +/- short deletion males and 3 GHRP1 +/- long deletion males. Will keep allof the GHR P1 +/- females (7 short deletion and 2 long deletion). These pigs will be bred (+/- x +/-) when they reach puberty to produce litters of +/+, +/-. and -/- piglets.
Impacts
What was accomplished under these goals?
We have completed the knockouts of the GHR promoters (both GHR P1 and GHR P2) and we are breeding the lines for data collection (phenotypes of the knockouts). This was the first objective of the grant. Completion of the first objective was delayed because of the COVID19 pandemic. Here is a summary of the pigs that we have on hand.Regardless of line (GHR P1 or GHR P2 KO), we plan to mate +/- females to +/- males to produce litters of +/+, +/-, -/- pigs. +/+ = wildtype; +/- = "het", one allele knocked out. -/- = "biallelic", both alleles knocked out. These litters of pigs will be used to make phenotypic measurements to determine the biology of the GHR promoters. GHR P2 KO pigs 131-1 (GHR P2 +/-) clone sow. This pig is 104 days since her last litter (12-7-2022) and 589 days old (b 8-9-2021). 162-1 (GHR P2 -/-) clone boar. This pig is 378 days old (b 3-8-2022). This boar has a dwarf phenotype. It appears to be fertile based on tests of in vitro fertilization. The conclusion is that GHR P2 may be essential for normal growth. 37-4 (GHR P2 +/-) gilt: This pig is319 days old (b 5-26-2022). 37-5 (GHR P2 +/-) 37-8 (GHR P2 +/-) boars. These pigs is 319 days old (b 5-26-2022). 126-3 (GH RP2 +/- boar). This pig is 104 days old (b 12-7-22). Wildtype gilts bred to 162-1 (GHR P2 -/-) clone boar. Should return to estrus this week or confirmed pregnant next week. Progency of these pigs will be ued for breeding teh GHR P2 KO lines. GHR P1 lines: There are two KO alleles (GHRP1 long and GHRP1 short) 123-1 and 123-3 (GHR P1 +/-; short allele) clone boars . These pigs are 110 days old (b 12-1-2022). P316, P320, P323 (GHRP1 -/-; short/long allele) clone sows . These pigs are 320 days old (b 4-5-2022). Farrowed 2-18-2023 (P316), 2-19-2023 (P323) and 2-20-2023 (P320). Weaned 3-16-2023. They appear to be normal size and fertile based litters. No obvious phenotype for these GHR P1 -/- pigs. P316, P320, and P323 piglets. These piglets are GHRP1 +/-; some with short deletion, others with long deletion).There are 15 males and 9 females.
Publications
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Progress 04/15/21 to 04/14/22
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Our primary activity for the next reporting period will be the propagation and establishment of lines of GHR P1 KO, GHR P2 KO, and control pigs. We will mate the cloned pigs that we have created to farrow pigs that are either homozygous or heterozygous for the KO or control. We plan to initiate studies of animal growth (Study 2A from the proposal). These will include measurements of body weight, skeletal structure, composition (back fat and loin eye area), and endocrinology (GH, GHBP, IGF1, and IGFBP). We will also test fertility and subsequent litter performance. Assuming that there are adequate numbers of pigs per line, we will collect tissue from a subset of founder animals to verify that the gene editing procedure gave expected KO of GHR 1A and GHR 1B-1C mRNA. In the meantime, we plan to study the GHR mRNA variants in the cell lines that we have created and a small number of KO pigs that have died. RNA will be extracted and submitted for RNA sequencing. GHR sequence will be aligned with the GHR genes to determine the effect of the GHR P1 and P2 KO on GHR mRNA transcripts.
Impacts
What was accomplished under these goals?
The project timeline stated that we would have founder knockout (KO) animals (GHR P1 KO and GHR P2 KO) during year 2 and that these founder animals would be used to produce experimental animals. The COVID-19 pandemic slowed the bench work needed to complete this project. Many of the laboratory supply chains needed to do the proposed work including the supply of ovaries that are used for collection of oocytes for somatic cell nuclear transfer (SCNT) were disrupted. In the past year, we were able to generate one GHR P2 biallelic knockout female pig (born August 2021), one biallelic knockout P2 male pig (born March 2022) and eight biallelic knockout GHR P1 female pigs (born March 2022). We are currently working with a male cell line for the GHR P1 KO using the same guides that we used to create the female clones for GHR P1. The GHR P2 female was inseminated to a wildtype (WT) boar in February and is pregnant. She is due to farrow on May 26, 2022. The GHR P2 female will produce a litter of "hets" (one copy of KO gene and one copy of WT genes). These animals will be used to propagate the GHR P2 line. The GHR P2 male KO will be bred to the GHR P2 female KO when he reaches puberty. This mating will create GHR P2 KO homozygous pigs. The litter of 8 GHR P1 KO female pigs will be grown and inseminated to WT boars to begin the creation of the GHR P1 KO lines. We assume that we will be able to create a GHR P1 KO male. This male will be bred to the GHR P1 KO females. All pigs that we have created at this time are clones from cell lines and have limited utility in the study of animal growth because the cloning has a large effect on the biology including growth rates.
Publications
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Progress 04/15/20 to 04/14/21
Outputs
Target Audience:
Nothing Reported
Changes/Problems:The COVID-19 pandemic slowed the bench work needed to complete this project. The University was closed from April through August and labs were not fully staffed. Many of the laboratory supply chains needed to do the proposed work including the supply of ovaries that are used for collection of oocytes for somatic cell nuclear transfer (SCNT) were also disrupted. These issues have led to slower progress than what we originally proposed for this work. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?The plan tosuccessfully producetwolines offounder knockout (KO) animals (GHR P1 KO and GHR P2 KO) and these founder animals willbe used to produce experimental animals. We will perform preliminary testing of GHR mRNA expression. Tissue will be collected from a subset of founder animals to verify that the gene editing procedure gave expected KO of GHR 1A and GHR 1B-1C mRNA.We should not observe GHR 1A mRNA in liver or any other tissue in GHR P1 KO pigs. Likewise, we should not observe GHR 1B-1C mRNA in GHR P2 KO pigs. We plan to initiate studies of animal growth (Study 2A from the proposal). These will include measurements of body weight, skeletal structure, composition (back fat and loin eye area), and endocrinology (GH, GHBP, IGF1, and IGFBP).
Impacts
What was accomplished under these goals?
The project timeline stated that we would have founder knockout (KO) animals (GHR P1 KO and GHR P2 KO) during year 2 and that these founder animals would be used to produce experimental animals. The COVID-19 pandemic slowed the bench work needed to complete this project. The University was closed from April through August and labs were not fully staffed. Many of the laboratory supply chains needed to do the proposed work including the supply of ovaries that are used for collection of oocytes for somatic cell nuclear transfer (SCNT) were also disrupted. In the past year, we were able to develop and validate appropriate CRISPR guide RNA for the GHR P2 KO. We successfully created a biallelic KO for the GHR P2 KO in a female cell line and these cells were used for a round of SCNT in March 2021. We produced 213 activated oocytes and performed 2 embryo transfer surgeries. The recipient gilts did not return to estrus following the transfer but we have not confirmed pregnancies at this time. We will attempt another round of SCNT on April 22, 2022. We are currently working with a male cell line for the GHR P2 KO using the same guides that we used to create the female cell line. The first round of transfections yielded 400 colonies. We did not get any biallelic edits but we did get about 20 heterozygous (het) colonies, 16 of which are growing well and being propagated. The het colonies will be frozen down and used if we are unable to isolate any GHR P2 biallelic edits for the male cell line. In addition to work on the GHR P2 KO, we are initiating work on the GHR P1 KO. This work will require the same series of experiments that we conducted for the GHR P2 KO. Specifically, we will develop primer pairs, buffer systems, and annealing temperatures that can be used to identify deletions and validate the guides that will be used in the creation of GHR P1 KO pigs.
Publications
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Progress 04/15/19 to 04/14/20
Outputs
Target Audience:
Nothing Reported
Changes/Problems:There was a slight delay in hiring the research specialist and an additional delay in the development of the GHR P2 deletion assay but these issues are now resolved and we are moving on to the creation of founder animals as described in the proposal. As of March 25, 2020, labs are shut down at the University (COVID 19) and only essential work (care and feeding of laboratory and farm animals) is being conducted. We do not know when labs will reopen but will resume work on the project when they do. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We plan to complete the work on the PCR-based assays to detect GHR P1 and GHR P2 gene deletions and validate the guide RNAs that will be used. Guides and Cas9 RNA will be injected into presumptive zygotes and the zygotes will be cultured to the blastocyst stage in vitro and surgically transferred to surrogates. All piglets produced from injected zygotes will be screened by amplimer size for obvious mono- and bi-allelic modification. The resulting PCR products will also be cloned and a minimum of 20 individual clones will be sequenced from each animal to determine the exact nature/sequence of each allele. We will then perform preliminary testing of GHR mRNA expression. We should not observe GHR 1A mRNA in liver or any other tissue in GHR P1 KO pigs. Likewise, we should not observe GHR 1B-1C mRNA in GHR P2 KO pigs. Tissue will be collected from a subset of founder animals to verify that the gene editing procedure gave expected knockout of GHR 1A and GHR 1B-1C mRNA. Founders with the most useful edits will be propagated.
Impacts
What was accomplished under these goals?
The proposed timeline states that founder animals will be generated during year 1. The award officially started on April 15, 2019. Most of the work for year 1 was to be performed by a research specialist working 50% time on the project. We identified an individual with appropriate qualifications but this individual could not begin work until August 2019 so this created a slight delay in getting the work started. We identified a 10 kb region containing P2 and exons 1B and 1C for targeting as outlined in the proposal.Guides were developed to target the sequence. We then began developing a PCR-based assay so that deletions could be detected with a sensitivity of less than 8 molecules. The development of the PCR-based assay created additional delays but this assay is essential so that we can evaluate the function of guide RNA to be used with Cas9. Guides that demonstrate an efficiency of gene modification of at least 25% will be used to produce embryos for embryo transfer. Design, evaluation, and use of CRISPR guide RNAs are routine at the MU National Swine Resource and Research Center but the DNA sequence within the P2 region contains many repetitive elements and offers very few unique sites for the placement of PCR primers. Through many iterations of primer pairs, buffer systems and annealing temperatures, we have now successfully developed a suitable PCR-based assay that can be used to identify deletions and validate the guides that will be used in the creation of the GHR P2 KO pigs. We plan to validate the guides and move directly to the creation of the GHR P2 pigs. The region surrounding GHR P1 and GHR 1A contains fewer repetitive elements and we expect that the development of the PCR-based assay to detect deletions will pose fewer problems. We will begin developing this assay while we begin to make the GHR P2 KO pigs. Creation of the GHR P1 lines will be initiated while we are making the GHR P2 lines.
Publications
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