PSI-7 Creation of gene-edited pigs for functional analysis of growth hormone receptor promoters in swine

Abstract

Abstract Growth hormone (GH; somatotropin) has a central role in animal growth, reproduction, and lactation. The biological actions of GH are dependent on the GH receptor (GHR) that is found in important target tissues such as liver, muscle, adipose tissue, and bone. It is widely accepted that GH is the primary hormone controlling animal growth and that its actions are mediated by the GHR. In humans, laboratory species, and farm animals there are two promoter complexes that control GHR expression. The first promoter complex (P1) controls the liver-specific inducible expression of the GHR1A mRNA. The second promoter complex (P2) controls the non-tissue specific constitutive expression of GHR1B mRNA. Despite the central role that GH and the GHR have in animal growth, the independent function of each promoter complex has not yet been tested in any species. The objective was to perform a germ-line deletion of each GHR promoter complex to test their function in a porcine model. The CRISPR/Cas9 system was used to delete the P1 and P2 promoter complexes in porcine fetal fibroblasts and cloned to create two independent lines for each promoter deletion. Adult animals that were heterozygous (het) for the GHRP1 and GHRP2 deletions were mated to create litters of +/+ (wildtype, WT), +/- (het), and -/- (knockout, KO) pigs for the respective promoters. An RTPCR of liver and muscle confirmed that deletion of GHRP1 and GHRP2 effectively knocked out GHR1A and GHR1B mRNA. Piglets were weighed at birth and approximately monthly until 2 (GHRP1) or 5 (GHRP2) mo of age. Piglets from the GHRP1 line 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) for GHRP1 piglets. Piglets from the GHRP2 line 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) but GHRP2-/- pigs weighed less (P < 0.01) at weaning compared with GHRP2+/+ or GHRP2+/- piglets (6.4 ± 0.8, 6.5 ± 0.4, and 4.5 ± 0.4 kg, respectively). Monthly BW for the GHRP2-/- piglets were less (P < 0.001) when compared with GHRP2+/+ or GHRP2+/- piglets. At 5 mo of age, the GHRP2-/- piglets were approximately 50% of the BW of GHRP2+/+ or GHRP2+/- (89.6 ± 6.7, 89.6 ± 3.9, and 43.1 ± 3.2 kg, respectively). In conclusion, the deletion of GHRP2 (and GHR1B mRNA) had a profound effect on pig growth. The GHRP1 deletion (GHR1A mRNA) did not affect growth with the caveat that the GHRP1 litters had not reached maturity. This work was supported by USDA NIFA 2019-67015-29484.